Covalent modifications of histone proteins, essential regulators of the activity of genes in eukaryotic cells, remodel the chromatin structure via a variety of enzymatic reactions. The reversible processes of some modifications, such as acetylation and deacetylation, are well characterized. Whether methylation and demethylation reversibly contribute to gene regulation, however, remains controversial. Recent studies have shown that methylation and demethylation are universally used to posttranslationally modify histones for the regulation of gene activity. In addition to LSD1, a nuclear amine oxidase homolog, which was found to function as a histone lysine demethylase, we and others found that some JmjC domain-containing proteins are histone demethylases. Specifically, we found that members of the JMJD2 protein family are histone demethylases that act on trimethyl groups of H3-K9 and H3-K36. Moreover, some members of this family also have activity for dimethyl groups. To understand the relationships between the structures and functions of these proteins, we have determined the structure of the catalytic core of the JMJD2A protein. From this structure, several novel structure features were revealed, such as the novel JmjN domain, the JmjC domain, the C-terminal domain, and a zinc finger motif. These unique structural features create a potential catalytic center. The structure also revealed a characteristic signature motif, which includes structural determinants for cofactors such as Fe(II) and a-ketoglutarate, a hallmark of non-heme containing oxygenases. There, however, are major questions that remained to be answered, such as the structural basis of the recognition of the substrate peptides by the catalytic core, the structural determinants of the specificity for the methyl groups, the mechanisms that regulate the activity of the enzyme, and the relationships among different JmjC domain-containing histone demethylases. Furthermore, only a small percentage of JmjC domain-containing proteins have been characterized. We believe that there are uncharacterized histone demethylases within the family of JmjC domain-containing proteins. Structural information derived from JMJD2A should be helpful in revealing these new histone demethylases. To answer these questions and to understand how the JmjC domain-containing histone demethylases function, three specific aims are proposed. Specific aim 1 - To characterize the structures and functions of the JMJD2 family members and to identify new histone demethylases;Specific aim 2 - To determine the high-resolution structures of the catalytic core of JMJD2A in the presence of different substrate peptides;Specific aim 3 - To determine the high-resolution structures of the entire JMJD2A protein and of the catalytic cores of other JmjC domain-containing proteins and to reveal the mechanisms that regulate the activity of JMJD2A and other related proteins.